1. Field of the Invention
This invention relates to the biodegradation capability of neutralized organophosphorus nerve agents. The invention further relates to biodegradation of hydrolyzed nerve agents using enzymes derived from bacteria.
2. Brief Description of Related Art
Neurotoxic chemical warfare (CW) agents, particularly G-type agents: GB, GD, and GF and V-type agents: VX and O-isobutyl S-(2-diethyl-aminoethyl) methylphosphonothioate (Russian-VX or R-VX) are in the stockpiles of the U.S. and former Solviet Union (Chapalamadugu, (1992), Microbiological and biotechnological aspects of metabolism of carbamates and organophosphates, (Crit. Rev. Biotechnol 12, 357-89, Fedorov (1994) Chemical Weapons in Russia: History, Ecology, Politics [Khimicheskoye Oruzhiye Vrossi: Istoriya, Ekologiya, Politka. Center of Ecological Policy of Russia, Moscow, Russia, Marrs et al., (1996) Chemical Warfare Agents: Toxicology and Treatments, New York, Somani (1992) Toxicodynamics of nerve agents, In Chemical Warfare Agents, pp. 67-123, Academic Press, New York). The USA signed the Chemical Weapons Convention Agreement (UN, 1992) in 1993 and ratified it on Apr. 25, 1997 that requires the destruction of all CW agents within ten years of ratification. Current chemical decontaminants contain corrosives, such as for example alkali agents (Yang, (1992), Decontamination of chemical warfare agents, Chem. Rev. 92, 1729-1743) and incineration has met with community opposition. The U.S. Army has been pursuing alternative technologies, such as enzymatic decontamination, that are safe and environmentally friendly (DeFrank (1993) Screening of halophilic bacteria and Alteromonas species for organophosphorus hydrolyzing enzyme activity, Chem. Boil. Interact. 87, 141-148). Degradation of G- and V-type agents can be accomplished using phosphoric triester hydrolase enzymes, e.g. organophosphorus hydrolase OPH (Dumas, (1989) Purification and properties of the phosphotriesterase from Pseudomonas diminuta, J. Biol. Chem, 264, 19659-19665, Dumas (1990) Inactivation of organophosphorus nerve agents by the phosphotriesterase from Pseudomonas diminuta, Arch. Biochem. Biophys, 277, 155-159.) and organophosphorus acid anhydrolase OPAA (Cheng, (1993) Purification and properties of a highly active organophosphorus acid anhydrolase from Alteromonas undina, Appl Environ Micrbiol., 59, 3138-3140, DeFrank, (1991) Purification and properties of an organophosphorus acid anhydrase from a halophilic bacterial isolate, J. Bacteriol 173, 1938-1943; Elashvili (1999), Purification and characterization of DFPase from Alteromonas haloplanktis ATCC 14393, Proceedings of the 1998 U.S. Army Edgewood Research, Development and Engineering Center Scientific Conference on Chemical and Biological Defense Research., ECBC-SP-004, pp 763-771), which, similar to alkali remove labile leaving moieties resulting in ionic methylphosphonate ester products. This enzymatic degradation process for the five selected nerve agents GB, GD, GF, VX, and R-VX would result in h-GB (IMPn), h-GD (PMPn), h-GF (CMPn), h-VX (EMPn), and h-R-VX (iBMPn) products, respectively (Scheme 1). 
The neurotoxic chemical warfare agents considered and shown have X as fluorine or other group that is more labile to hydrolysis than the A alkyl group and therefore is designated as the leaving group.
Although the invention is useful for decontaminating nerve agents that are in stock piles, it is also useful in the field for nerve agent decontamination of affected areas.
There is no suitable known method for further degrading h-agents into hydrolyzed agents. The hydrolysis of V- and G-type agents by alkali (e.g. NaOH) or by enzymes (e.g. OPH or OPAA) generates stable Alkyl methylphosphonate products, which could inhibit the activity of the initial agent hydrolase enzyme. For example, diisopropyl phosphate, a product of the diisopropyl fluorophosphate hydrolysis by OPH, was shown to be inhibitory to the OPH activity (Elashvili (1999), Purification and characterization of DFPase from Alteromonas haloplanktis ATCC 14393, Proceedings of the 1998 U.S. Army Edgewood Research, Development and Engineering Center Scientific Conference on Chemical and Biological Defense Research, ECBC-SP-004, pp 763-771). PEH hydrolysis of Alkyl methylphosphonates, which are persistent in the environment, to the biodegradable methylphosphonate product that is easily degraded by environmentally prevalent microbes utilizing it as a phosphorus source, not only results in the enhancement of the effectiveness of the agents degradation, but also leads to their subsequent mineralization by common soil and aquatic bacteria in the environment. Therefore, there is a present need to further degrade these chemicals.
It is an object of the present invention to provide a method of biodegrading nerve agents.
It is another object of the present invention to biodegrade nerve agents with enzymatic agents.
It is still another object of the present invention to further destruction of nerve agents using microorganisms that produce enzymes that degrade hydrolyzed nerve agents.
These and other objects of the present invention will become apparent upon further reading of this disclosure.